Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C–C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with α,β-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of α,β-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten α,β-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic α,β-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an R-selectivity of 83% ee. The best substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with R-selectivity >99.9% ee. Michaelis–Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover kcat of 0.71 ± 0.03 s–1 and a Km of 2.46 ± 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 Å resolution was obtained, and docking studies showed a productive pose with the substrate.@en

Despite the increasing demand for efficient and sustainable chemical processes, the development of scalable systems using biocatalysis for fine chemical production remains a significant challenge. We have developed a scalable flow system using immobilized enzymes to facilitate flavin-dependent biocatalysis, targeting as a proof-of-concept asymmetric alkene reduction. The system integrates a flavin-dependent Old Yellow Enzyme (OYE) and a soluble hydrogenase to enable H2-driven regeneration of the OYE cofactor FMNH2. Molecular hydrogen was produced by water electrolysis using a proton exchange membrane (PEM) electrolyzer and introduced into the flow system via a designed gas membrane addition module at a high diffusion rate. The flow system shows remarkable stability and reusability, consistently achieving >99% conversion of ketoisophorone to levodione. It also demonstrates versatility and selectivity in reducing various cyclic enones and can be extended to further flavin-based biocatalytic approaches and gas-dependent reactions. This electro-driven continuous flow system, therefore, has significant potential for advancing sustainable processes in fine chemical synthesis.@en

Ene-reductases from the old yellow enzyme (OYE) family have been traditionally employed in the reduction of conjugated C═C double bonds. This study explores the underutilized oxidative potential of OYEs, demonstrating their capability to catalyze the enantioselective desaturation of carbonyl compounds. Utilizing a deprotonated tyrosine residue as a catalytic base, we developed a method to enable OYE-catalyzed desaturation at ambient temperature and alkaline pH without the need for high-temperature conditions. Through screening of various OYE enzymes, we identified several candidates from different genera with enhanced desaturase activity across different substrates. This work broadens the scope of biocatalytic applications for OYEs, introducing a novel approach to the synthesis of chiral α,β-unsaturated carbonyl compounds.@en

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN⁺). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN⁺ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN⁺ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA⁺), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)⁺.

@en